Article

pubs.acs.org/JAFC

Insecticidal and Acetylcholine Esterase Inhibition Activity of

Apiaceae Plant Essential Oils and Their Constituents against Adults
of German Cockroach (Blattella germanica)
Hwa-Jeong Yeom, Jae Soon Kang, Gil-Hah Kim, and Il-Kwon Park*,

Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul 130-712, Republic of Korea

Department of Plant Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea

ABSTRACT: We evaluated the insecticidal and acetylcholine esterase (AChE) inhibition activity of 11 Apiaceae plant essential
oils and their constituents in adult male and female Blattella germanica. Of the 11 Apiaceae plant essential oils tested, dill
(Anethum graveolens), carvi (Carum carvi), and cumin (Cuminum cyminum) demonstrated >90% fumigant toxicity against adult
male German cockroaches at a concentration of 5 mg/lter paper. In a contact toxicity test, dill (Anethum graveolens), carvi
(Carum carvi), cumin (Cuminum cyminum), and ajowan (Trachyspermum ammi) produced strong insecticidal activity against
adult male and female German cockroaches. Among the test compounds, (S)-(+)-carvone, 1,8-cineole, trans-dihydrocarvone,
cuminaldehyde, trans-anethole, p-cymene, and -terpinene demonstrated strong fumigant toxicity against adult male and female
B. germanica. In a contact toxicity test, carveol, cuminaldehyde, (S)-(+)-carvone, trans-anethole, thymol, and p-cymene showed
strong contact toxicity against adult male and female B. germanica. IC50 values of -pinene, carvacrol, and dihydrocarvone against
female AChE were 0.28, 0.17, and 0.78 mg/mL, respectively. The toxicity of the blends of constituents identied in 4 active oils
indicated that carvone, cuminaldehyde, and thymol were major contributors to the fumigant activity or contact toxicity of the
articial blend.
KEYWORDS: Apiaceae plant essential oils, fumigant toxicity, contact toxicity, German cockroach, acetylcholine esterase inhibition

INTRODUCTION
The German cockroach, Blattella germanica (L.) (Dictyoptera:
MATERIALS AND METHODS
Plant Essential Oils and Chemicals. Essential oils of ammi
Blattellidae), is a small cockroach about 1.3 cm (0.51 in) to 1.6 visnaga (Ammi visnaga), celery (Apium graveolens), pastinak (Pastinaca
cm (0.63 in) in length, and is commonly found in houses, sativa), and parsely (Petroselinum sativum) were purchased from
schools, hospitals, and other large buildings. They are Oshadhi (Weinstrasse, Buhl/Baden, Germany). Dill (Anethum grave-
considered important indicators of hygiene, because they can olens), carvi (Carum carvi), coriander (Coriandrum sativum), carrotseed
(Daucus carota), cumin (Cuminum cyminum), galbanum (Ferula
cause allergic reactions in sensitive people,1 and transmit several
galbanif lua), and ajowan (Trachryspermum ammi) were purchased
human pathogens such as viruses, bacteria, protozoa, and from Jinarome (USA). These plant essential oils are listed in Table 1.
helminthes.2 Control of German cockroaches is primarily Carveol (purity, 97%), (S)-(+)-carvone (96%), 1,8-cineole (99%),
dependent on continued applications of residual insecticides, (+)-limonene (97%), myrcene (95%), cuminaldehyde (98%), trans-
such as chlorpyrifos, DDVP, pyrethroids, and bendiocarb.3
However, their repeated use has resulted in the development of Table 1. List of Apiaceae Plant Essential Oils Tested
resistance and has caused serious human health concerns.1,4
Because of the many side eects of these conventional oil scientic name region part
pesticides, the development of new and safe German cockroach ammi visnaga Ammi visnaga Morocco owering plant
control agents is essential.5,6 dill Anethum graveolens Bulgaria seeds
Plant essential oils are good candidates as German cockroach celery Apium graveolens France seeds
control agents. They can be easily extracted by steam- carvi Carum carvi Egypt seeds
distillation, and they consist of mixtures of many bioactive coriander Coriandrum sativum Russia herb
compounds, such as alcohols, aldehydes, ketones, esters, cumin Cuminum cyminum Egypt seeds
aromatic phenols, and lactones as well as monoterpenes and carrotseed Daucus carota France seeds
sesquiterpenes.7 Many essential oils and their constituents galbanum Ferula galbanif lua Iran resin
demonstrate insecticidal or repellent activity against the pastinak Pastinaca sativa Croatia whole plant
German cockroach.812 Furthermore, plant essential oils are parsely Petroselinum sativum Hungary herb
highly volatile and there is little concern regarding their residue ajowan Trachyspermum ammi India seeds
in the eld and water.13,14 In this study, we investigated the
insecticidal and acetylcholine esterase (AChE) inhibition Received: May 8, 2012
activities of plant essential oils and their components against Revised: June 26, 2012
the German cockroach in order to nd potential alternatives to Accepted: July 2, 2012
current insecticides and their mode of action. Published: July 3, 2012

2012 American Chemical Society 7194 dx.doi.org/10.1021/jf302009w | J. Agric. Food Chem. 2012, 60, 71947203
Journal of Agricultural and Food Chemistry Article

Figure 1. Fumigant toxicity of the oil, full mixture, and selected blends of the constituents of dill, carvi, and cumin oils in German cockroach male
adults. The concentration of dill and carvi oil was 20 mg/lter paper. The concentrations of the full mixture of dill and carvi oils were 15.82 mg/lter
paper and 16.78 mg/lter paper, respectively. The concentrations of cumin oil and the full mixture were 5 mg/lter paper and 3.82 mg/lter paper,
respectively. The concentrations of other blends were determined by removing each constituent equivalent to the ratio identied in dill, carvi, and
cumin oils. Mean values corresponding to each treatment with dierent letters are signicantly dierent from each other (dill oil, F10,33 = 37.12, p <
0.0001; carvi oil, F5,18 = 15.27, p < 0.0001; cumin oil, F15,48 = 120.31, p < 0.0001, Schees test).

anethole, and dihydrocarvone (98%) were obtained from Sigma-
Aldrich (Milwaukee, WI). p-Cymene (95%), -terpinene (97%), -
terpinene (85%), terpinen-4-ol (99%), menthol (99%), linalool oxide
(97%), and thymol (99%) were purchased from Fluka (Buchs,
Switzerland). (+)--Pinene (95%), bornyl acetate (70%), -pinene
(94%), carvacrol (95%), and -phellandrene (65%) were obtained
from Tokyo Kasei (Tokyo, Japan). Acetone was purchased from
Merck (99.8%), and neral (98%) was synthesized in the laboratory.
Insect. B. germanica was cultured in the laboratory without
exposure to any insecticide. The cockroaches were provided water
from a glass ask tted with a cotton stopper and dried mouse food.
The cockroaches were maintained at 27 1 C and 60% RH under a
16:8 h light:dark cycle.
Gas Chromatography. Gas chromatography (GC) analysis was
performed using Agilent 6890N (Santa Clara, CA, USA) equipped
with a ame ionization detector (FID). Retention times, for
comparison with those of authentic compounds, were measured
using DB-1MS and HP-INNOWAX columns (internal diameter [i.d.],
30 m 0.25 mm; lm thickness, 0.25 m; J&W Scientic, Folsom,
CA, USA). The oven temperature was programmed as isothermal at
40 C for 1 min, then raised to 250 at 6 C/min, and held at this
temperature for 4 min. Helium was used as the carrier gas at the rate of
1.5 mL/min. For chiral GC separation of carvone, a Beta DEX 225
(i.d., 30 m 0.25 mm; lm thickness, 0.25 m; Supelco) was used.
The temperature program was as follows: 130 C for 10 min and then
increased to 200 C at a rate of 10 C/min. The carrier gas had a ow
rate of 1.0 mL/min. Retention indices were determined in relation to a
homologous series of n-alkanes (C7C20), (C8C22) under the same
operating conditions. Further identication was made by enhancing
the integrated area by coinjection with oil and authentic samples.
Gas ChromatographyMass Spectrometry. Essential oils were
analyzed using a gas chromatograph (Agilent 7890A)mass
spectrometer (Agilent 5975C MSD) (GCMS) (Santa Clara, CA,
USA) equipped with a DB-5MS column (i.d., 30 m 0.25 mm; lm
thickness, 0.25 m; J&W Scientic, Folsom, CA, USA). The oven
temperature was programmed as in GC-FID analysis. Helium was used
as the carrier gas at the rate of 1.0 mL/min. An euent of the GC
column was introduced directly into the source of the MS via a transfer
line (250 C). Ionization was achieved using an electron impacter (70
eV; source temperature, 230 C). The scan range was 41400 amu.
Most components of the oil were tentatively identied by comparing
the mass spectra of each peak with those of authentic samples in the
NIST MS library.
Fumigant Toxicity Test. A paper disk (8 mm, Advantec) treated
with the essential oil or compound being tested was placed in the
bottom lid of a glass cylinder (diameter, 9.5 cm; height, 19 cm) with a
wire sieve tted 9.5 cm above the bottom; thereafter, the lid was sealed
with Paralm (Pechiney Plastic Packaging Company, Chicago, USA).
Ten adult male and female German cockroaches were placed on the
sieve. This prevented direct contact of the cockroach with the test
plant oils and compounds. The insects were maintained at 25 1 C
and 60% RH. The adult cockroaches were considered dead if their
appendages did not move when prodded with a brush. Cumulative
mortalities were determined 48 h after treatment. All treatments were
replicated 4 times.
Contact Toxicity Test. Appropriate doses of the test compounds
dissolved in acetone were topically applied to the thorax of the adult
male and female German cockroaches (anesthetized using CO2) with a
microapplicator (Burkard, Hertfordshire, U.K.). The controls received
acetone (1 L). Batches of 10 treated adults were put into a Petri dish

7195 dx.doi.org/10.1021/jf302009w | J. Agric. Food Chem. 2012, 60, 71947203

Journal of Agricultural and Food Chemistry Article

Figure 2. Contact toxicity of the oil, full mixture, and selected blends of the constituents of dill, carvi, cumin, and ajowan oils in female adult German
cockroaches. The concentration of dill, carvi, and ajowan oils was 2 mg/. The concentration of cumin oils was 1 mg/. The concentrations of
articial mixtures of dill, carvi, and ajowan oils were 1.58, 1.66, and 1.96 mg/, respectively. The concentration of articial mixtures of cumin oil was
0.76 mg/. The concentrations of other blends were determined by removing each constituent equivalent to the ratio identied in dill, carvi, cumin,
and ajowan oils. Mean values corresponding to each treatment with dierent letters are signicantly dierent from each other (dill oil, F10,44 = 95.45,
p < 0.0001; carvi oil, F5,248 = 8.33, p < 0.0001; cumin oil, F15,64 = 88.75; ajowan oil, F13,564 = 47.85 p < 0.0001, Schees test).

(diameter, 9.5 cm; height, 2 cm). Mortality was determined 48 h after
treatment. Each assay was performed 5 times.
Comparative Toxicities. To determine the contribution of each
constituent to fumigant or contact toxicity against German
cockroaches, we prepared blends of all constituents for 4 active oils
that mimicked the natural oils. We also prepared a number of blends,
each lacking 1 constituent (Figures 1 and 2). Blends were based on the
natural composition of the 4 active oils, as indicated by GC-FID
(Table 4). In a fumigant toxicity test, the concentration of dill and
carvi oils was 20 mg/lter paper. The concentrations of the full
mixture of dill and carvi oils were 15.82 mg/lter paper and 16.78 mg/
lter paper, respectively. The concentrations of cumin oil and the full
mixture were 5 mg/lter paper and 3.82 mg/lter paper, respectively.
The concentrations of other blends were determined by removing
each constituent equivalent to the ratio identied in dill, carvi, and
cumin oils. In the contact toxicity test, the concentration of dill, carvi,
and ajowan oil was 2 mg/. The concentration of cumin oils was 1
mg/. The concentrations of articial mixtures of dill, carvi, and
ajowan oils were 1.58, 1.66, and 1.96 mg/, respectively. The
concentration of articial mixtures of cumin oil was 0.76 mg/. The
concentrations of other blends were determined by removing each
constituent equivalent to the ratio identied in dill, carvi, cumin, and
ajowan oils. We also compared the toxicities of the complete and
incomplete blends with those of the pure 4 active oils.
Primary AChE Inhibition Assay and IC50 Estimation. To
extract crude protein, an adult cockroach was ground using a glass
tissue-grinder (Wheaton Industries Inc., Millville, NJ) in protein
extraction buer (0.1 M Tris-HCl containing 0.02 M NaCl and 0.5%
Triton X-100, pH 7.8) and protease inhibitor cocktail (Sigma-Aldrich,
St. Louis, MO). The ground cockroach was centrifuged at 15,000 rpm
for 15 min, and the crude protein was collected from the ground
mixture. All procedures to extract the crude protein were performed
under 4 C. The AChE activity was measured using the modied
Ellman method.15 All chemicals tested were diluted to 100 mg/mL in
acetone (carvacrol and thymol were diluted to 50 mg/mL). Then, 2
L of the chemicals (nal concentration was 0.5 mg/mL or 1 mg/mL)
and 30 L of crude protein were mixed in a 96-well microplate
containing 148 L of protein extraction buer. Next, the mixture was
incubated for 10 min at room temperature and then acetylthiocholine
iodide (nal concentration, 1 mM) and 5,5-dithiobis(2-nitrobenzoic
acid) (nal concentration, 0.4 mM) were added. The control was
treated by adding acetone without other chemicals. The enzyme
activity was measured for 30 min at 1 min intervals at 405 nm and rt.
The inhibition rate of the treatment against control was calculated in
percentage by using the following formula:
% inhibition rate = 100 (enzyme activity of treatment
/enzyme activity of control 100)
The primary AChE inhibition assay was replicated at least 3 times.
Three chemicals-pinene, carvacrol, and dihydrocarvonewere
selected to determine IC50 values because of their higher inhibition
rate. The following concentrations of -pinene, carvacrol, and
dihydrocarvone were used: 2, 1, 0.5, 0.1, and 0.05 mg/mL. All
treatments were replicated 3 times at each concentration. The enzyme
activity was measured as described above, and the IC50 was estimated
using probit analysis.16
Statistical Analysis. The percentage of mortality and primary
AChE inhibition rate was determined and transformed to arcsine
square-root values for analysis of variance. Treatment mean values
were compared and analyzed using Schees test.16 Mean (SE)
values of untransformed data have been reported.

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Table 2. Fumigant Toxicity of 11 Apiaceae Plant Essential Oils against Male and Female Adults of German Cockroach
mortality (%, mean SE, N = 40)
male female
a
plant essential oils 20 10 5 2.5 1.25 20 10
ammi visnaga 17.5 4.8 bb 0c c 2.5 2.5 d
dill 100 a 100 a 100 a 27.5 7.5 b 2.5 2.5 a 95.0 5.0 a 7.5 4.8 bc
celery 12.5 4.8 bc 2.5 2.5 d
carvi 100 a 100 a 100 a 60.0 4.0 a 2.5 2.5 a 97.5 2.5 a 20 ab
coriander 95.0 2.9 a 12.5 4.8 bc 37.5 4.8 c 5.5 5.0 bc
cumin 100 a 100 a 90.0 7.0 a 2.5 2.5 c 87.5 6.3 ab 0c
carrotseed 10 bc 0d
galbanum 15.0 2.9 bc 10.0 4.1 d
pastinak 2.5 2.5 bc 0d
parsely 97.5 2.5 a 7.5 4.8 bc 15.0 2.9 cd
ajowan 100 a 17.5 2.5 b 65.0 2.9 b 32.5 4.8 a
control 0c 0c 0b 0c 0a 0d 0c
F11,36 = 25.0 F7,24 = 26.04 F3,12 = 50.0 F3,12 = 79.17 F2,9 = 16.67 F11,36 = 46.5 F5,18 = 47.2
p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p = 0.6224 p < 0.0001 p < 0.0001
a
mg/lter paper. bMeans within a column followed by the same letters are not signicantly dierent (Schees test). cNot tested.

Table 3. Contact Toxicity of 11 Apiaceae Plant Essential Oils against Male and Female Adults of German Cockroach
mortality (%, mean SE, N = 50)
male female
a
plant essential oils 2 1 0.5 0.25 2 1
ammi visnaga 98.0 2.0 ab 10.0 3.1 cd c 12.0 4.9 d
dill 100 a 100 a 62.0 3.7 ab 6.0 4.0 b 84.0 5.0 ab 20.0 3.2 cd
celery 100 a 74.0 5.1 b 4.0 2.4 d 96.0 4.0 a 22.0 5.8 cd
carvi 100 a 100 a 20.0 5.5 cd 96.0 4.0 a 50.0 5.5 ab
coriander 86.0 2.4 b 26.0 4.0 c 72.0 2.0 b 34.0 2.4 bc
cumin 100 a 100 a 42.0 5.8 bc 0b 100 a 64.0 2.4 a
carrotseed 100 a 100 a 18.0 8.0 cd 74.0 2.4 b 34.0 2.4 bc
galbanum 82.0 2.0 b 12.0 3.7 cd 40.0 3.1 c
pastinak 100 a 92.0 4.9 ab 8.0 3.7 d 64.0 2.4 b 10.0 3.2 d
parsely 100 a 24.0 6.0 c 66.0 2.4 b 8.0 2.0 d
ajowan 100 a 100 a 76.0 5.0 a 24.0 2.4 a 100 a 42.0 5.8 abc
control 0c 0d 0d 0b 0d 0d
F11,48 = 5.8 F11,48 = 52.5 F7,32 = 117.5 F3,16 = 27.5 F11,48 = 47.5 F9,40 = 70.0
p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001
a
mg/adult. bMeans within a column followed by the same letters are not signicantly dierent (Schees test). cNot tested.

RESULTS AND DISCUSSION

Fumigant and Contact Toxicities of the Plant
Essential Oils. When 11 plant essential oils were subjected
to bioassays, mortalities varied according to the oil type and
dose (Tables 2 and 3). In a test with adult male German
cockroaches, dill (Anethum graveolens), carvi (Carum carvi), and
cumin (Cuminum cyminum) essential oils showed 100%
fumigant toxicity at 20 mg/lter paper and 10 mg/lter
paper concentrations. At 5 mg/lter paper concentration, these
3 oils caused 90% mortality. Coriander and parsley oils caused
95% and 97.5% mortality at 20 mg/mL concentration, but the
mortality reduced to 12.5% and 7.5% at 10 mg/mL
concentration, respectively. The other oils showed weak
fumigant toxicity at 20 mg/lter paper concentration. In a
contact toxicity test, dill, carvi, cumin, carrotseed, and ajowan
oils demonstrated 100% insecticidal activity against male
German cockroaches at 1 mg/ concentration. Cumin, ajowan,
celery, carvi, and dill oils demonstrated >80% contact toxicity
against female adults at 2 mg/ concentration. The other oils
showed moderate or weak activity. The insecticidal or
7197
nematicidal activity of dill, carvi, cumin, and ajowan has been
reported in previous studies,1719 but there have not been any
reports about their fumigant and contact toxicities toward the
German cockroach.
Chemical Components of the Plant Essential Oils. The
chemical compositions of dill, carvi, cumin, and ajowan
essential oils are shown in Table 4. The chemical compositions
of ajowan, carvi, dill, and cumin oils have been reported in our
previous study.18,19 There was only a little dierence in the
composition rate of the constituents of ajowan, carvi, and dill
oils. However, 5 compoundslimonene, cis-linalool oxide,
menthol, neral, and bornyl acetatewere newly identied in
cumin oil, as compared to the results of our previous study.18
The main components of dill oil were -pinene (0.37%), -
myrcene (0.17%), -phellandrene (3.90%), p-cymene (3.05%),
1,8-cineole (1.20%), limonene (22.83%), dill ether (5.04%),
trans-dihydrocarvone (0.9%), cis-dihydrocarvone (0.91%), and
carvone (40.77%). Limonene (27.01%), cis-carveol (0.52%),
trans-carveol (0.39%), and carvone (40.77%) were detected as
the main components in carvi oil. -Pinene (0.69%), -pinene
dx.doi.org/10.1021/jf302009w | J. Agric. Food Chem. 2012, 60, 71947203
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Table 4. Chemical Analysis of Dill, Carvi, Cumin, and Ajowan Essential Oils
retention index amount (w/w, %)
no. compound DB-1MS HP-Innowax dill carvi cumin ajowan
1 -pinene 929 1020 0.37 c
0.69 0.87
2 -pinene 969 1107 9.14 1.26
3 -myrcene 982 1165 0.17 0.53 0.48
4 -phellandrene 995 1165 3.9 0.39
5 -terpinene 1007 1181 0.13
6 p-cymene 1011 1273 3.05 26.88 24.4
7 1,8-cineole 1018 1209 1.2 0.32
8 limonene 1020 1200 22.83 27.01 0.62 0.44
9 -terpinene 1049 1247 16.31 27.77
10 cis-linalool oxide 1072 1450 0.69
11 menthol 1158 1643 0.58
12 terpinen-4-ol 1160 1611 0.32
13 dill ethera 1165 1484 5.04
14 trans-dihydrocarvoneb 1169 1611 0.9
15 cis-dihydrocarvoneb 1175 1631 0.91
16 cis-carveolb 1196 1848 0.52
17 trans-carveolb 1207 1879 0.39
18 cuminaldehyde 1210 1789 17.26
19 (S)-(+)-carvone 1213 1738 40.77 55.98
20 neral 1218 1690 0.41
21 trans-anethole 1259 1833 2.52
22 bornyl acetate 1267 1582 0.56
23 thymol 1273 2207 41.77
24 carvacrol 1278 2236 0.55
sum 79.14 83.9 76.58 98.31
a
Structure was tentatively identied by comparison of mass spectrum in the library. bMixture of cis- and trans-isomers was determined from the
literature report.33 cNot detected.

(9.14%), -myrcene (0.53%), -phellandrene (0.39%), p-
cymene (26.88%), limonene (0.62%), -terpinene (16.31%),
cis-linalool oxide (0.69%), menthol (0.58%), cuminaldehyde
(17.26%), neral (0.41%), trans-anethole (2.52%), and bornyl
acetate (0.56%) were identied as the major components of
cumin oil. The main components of ajowan oil were -pinene
(0.87%), -pinene (1.26%), -myrcene (0.48%), -terpinene
(0.13%), p-cymene (24.4%), 1,8-cineole (0.32%), limonene
(0.44%), -terpinene (27.77%), terpinen-4-ol (0.32%), thymol
(41.77%), and carvacrol (0.55%).
Fumigant and Contact Toxicities of the Individual
Compounds. The fumigant and contact toxicities of the
individual compounds from dill, carvi, cumin, and ajowan oils
are shown in Tables 5 and 6. In a fumigant toxicity test, carvone
demonstrated the strongest activity against male adults,
followed by trans-anethole, dihydrocarvone, 1,8-cineole, -
terpinene, and p-cymene. In a test with female adults, 1,8-
cineole showed the strongest insecticidal activity followed by
dihydrocarvone, carvone, trans-anethole, and cuminaldehyde. In
a contact toxicity test with male adults, trans-anethole and
thymol demonstrated the most potent activity. Insecticidal
activity of carvacrol was 84% at 0.25 mg/, but it reduced to
4% at 0.125 mg/. In a contact test with female adults, thymol
was the most toxic followed by trans-anethole, cuminaldehyde,
and carvacrol. However, toxicities of plant essential oils and
their components were weaker than those of conventional
insecticides such as chlorpyrifos (LD50 = 0.015 g/female) or
deltamethrin (LD50 = 0.0054 g/female).20 Philips et al.11 and
Phillips and Appel12 have already reported the fumigant and
contact toxicities of 12 essential oil components against
German cockroaches: carvacrol, 1,8-cineole, trans-cinnamalde-
7198
hyde, citornellic acid, eugenol, geraniol, limonene, linalool,
menthone, -pinene, -pinene, and thymol. They reported that
1,8-cineole was the most toxic to male and female adult
German cockroaches in a fumigant test. The fumigant toxicity
of carvacrol and thymol was less than that of -pinene, -
pinene, and limonene. However, thymol and carvacrol showed
strong contact toxicity against adult male and female German
cockroaches. This observation and our results conrm that
thymol and carvacrol showed insecticidal activity against
German cockroaches by direct contact, and not via fumigation.
In our fumigant toxicity test, mortality with -pinene, -pinene,
and limonene was not higher than that with the other
compounds. This result did not agree with the results of
Phillips and Appel.12 The dierence in the insecticidal activity
of these compounds might be because we used dierent strain
of German cockroach.
Comparative Toxicities of the Blends. A fumigant
toxicity test with articial mixtures showed that blends of dill,
carvi, and cumin containing 9, 4, and 13 known constituents of
the 3 oils were the most toxic (Figure 1). Insecticidal activity of
articial mixtures containing all the constituents did not dier
signicantly from that of the 3 essential oils (Figure 1; p <
0.0001). Component elimination assays of dill and carvi oils
indicated that the omission of carvone from the articial
mixture caused a signicant decrease in the fumigant toxicity of
the blend (dill oil: F10,33 = 37.12, p < 0.0001, carvi oil: F5,18 =
15.27, p < 0.0001). These results indicated that carvone is a
major contributor to the fumigant toxicity of dill and carvi oils.
Omission of other compounds from the articial mixture did
not cause a signicant dierence in the fumigant toxicity of the
blend. For cumin oil, component elimination assays (Figure 1)
dx.doi.org/10.1021/jf302009w | J. Agric. Food Chem. 2012, 60, 71947203
 Table 5. Fumigant Toxicity of Components from Dill, Carvi, and Cumin Essential Oils against Male and Female Adults of German Cockroach
mortality(%, mean SE, N = 40)
male female
compounds 20a 10 5 2.5 1.25 0.625 20 10 5 2.5 1.25
b c b
-pinene 65.0 2.9 bc 12.5 2.5 c 52.5 4.8 c 12.5 2.5 c
-pinene 57.5 4.8 c 12.5 6.3 c 57.5 4.8 bc 10.0 4.1 c
-myrcene 5.0 2.9 d 2.5 2.5 d
-phellandrene 100 a 57.5 2.5 b 2.5 2.5 cd 100 a 70.0 4.1 b 0d
p-cymene 100 a 100 a 12.5 4.8 cd 100 a 97.5 2.5 a 10.0 4.1 d
1,8-cineole 100 a 100 a 100 a 0d 100 a 100 a 100 a 100 a 0
limonene 85.0 6.5 ab 17.5 7.5 c 75.0 5.0 b 10.0 4.1 c
-terpinene 100 a 100 a 17.5 4.8 c 100 a 92.5 4.8 a 15.0 2.9 d
linalool oxide 15.0 5.0 d 2.5 2.5 d
menthol 2.5 2.5 d 2.5 2.5 d
dihydrocarvone 100 a 100 a 100 a 42.5 4.8 c 2.5 2.5 c 100 a 100 a 100 a 5.0 2.9 cd
Journal of Agricultural and Food Chemistry

carveol 2.5 2.5 d 2.5 2.5 d

cuminaldehyde 100 a 97.5 2.5 c 67.5 2.5 b 62.5 2.5 b 40 7.1 b 7.5 2.5 100 a 100 a 55.0 2.9 c 17.5 4.8 c
(S)-(+)-carvone 100 a 100 a 100 a 100 a 100 a 2.5 2.5 100 a 100 a 97.5 2.5 ab 77.5 4.8 b 0
neral 17.5 8.5 d 17.5 2.5 d
trans-anethole 100 a 100 a 100 a 77.5 4.8 b 7.5 2.5 c 100 a 100 a 82.5 4.8 b 15.0 2.9 cd
bornyl acetate 80 abc 65 2.8 b 10 4.0 cd 7.5 4.7 d
control 0d 0c 0d 0d 0c 0 0d 0c 0d 0d 0

7199
F17,54 = 42.5 F12,39 = 37.8 F9,30 = 30.0 F5,18 = 34.7 F4,15 = 50.0 F2,9 = 16.7 F17,54 = 28.2 F11,36 = 28.5 F8,27 = 27.8 F5,18 = 41.7
p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p = 0.0751 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001
a
mg/lter paper. bMeans within a column followed by the same letters are not signicantly dierent (Schees test). cNot tested.
Article

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Journal of Agricultural and Food Chemistry Article

Table 6. Contact Toxicity of Components from Dill, Carvi, Cumin, and Ajowan Essential Oils against Male and Female Adults
of German Cockroach
mortality (%, mean SE, N = 50)
male female
compounds 1a 0.5 0.25 0.125 1 0.5 0.25
-pinene 54.0 2.4 cdef b
38.0 5.8 defgh c
46.0 2.4 cdefg 10.0 4.5 d
-pinene 48.0 2.0 def 6.0 4.0 hi 12.0 3.7 hi
-myrcene 42.0 2.0 ef 18.0 3.7 fghi 34.0 4.0 efgh
-phellandrene 80.0 4.5 abc 34.0 2.4 defgh 0c 76.0 2.4 abc 2.0 2.0 d
-terpinene 68 5.8 bcde 26 5.0 efghi 42 3.7 defgh 26 5.0 cd
p-cymene 98.0 2.0 a 76.0 2.4 abc 0c 68.0 2.0 bcd 4.0 2.4 d
1,8-cineole 98.0 2.0 a 56.0 2.4 bcde 12.0 3.7 c 28.0 4.9 fghi 24.0 4.0 cd
limonene 40.0 5.5 f 20.0 3.2 fghi 26.0 5.1 fghi
-terpinene 90.0 0.0 ab 14.0 6.0 ghi 42.0 3.7 defgh 14.0 6.0 d
linalool oxide 96.0 2.4 a 60.0 4.5 bcd 4.0 2.4 c 20.0 6.3 fghij
menthol 52.0 3.7 def 16.0 4.0 fghi 18.0 3.7 ghi
dihydrocarvone 98.0 2.0 a 42.0 4.9 defg 2.0 2.0 c 50.0 3.2 cdef 12.0 2.0 d
carveol 96.0 4.0 a 80.0 3.2 ab 2.0 2.0 c 60.0 5.5 cde 20.0 4.5 cd
cuminaldehyde 100 a 96.0 2.4 a 70.0 3.2 b 0b 98.0 2.0 ab 90 a 6.0 2.4 b
carvone 100 a 80.0 5.5 ab 16.0 2.4 c 98.0 2.0 ab 42.0 3.7 bc 2.0 2.0 b
neral 80.0 4.5 abc 48.0 5.8 bcdef 6.0 4.0 c 22.0 3.7 fghi
trans-anethole 100 a 100 a 96.0 2.4 a 6.0 2.4 b 100 a 96.0 2.4 a 14.0 4.0 b
thymol 100 a 98.0 2.0 a 98.0 2.0 a 62.0 2.0 a 100 a 94.0 4.0 a 68.0 3.7 a
carvacrol 100 a 100 a 84.0 2.4 ab 4.0 4.0 b 94.0 2.4 ab 62.0 5.8 b 8.0 3.7 b
bornyl acetate 74 6.7 abcd 46 5.0 cdefg 2.0 2.0 c 46 6.7 cdefg 0d
control 0g 0i 0c 0b 0i 0d 0b
F20,84 = 50.4 F20,84 = 77.1 F13,56 = 28.5 F4,20 = 26.0 F20,84 = 69.5 F14,60 = 67.3 F5,24 = 45.0
p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001
a
mg/adult. bMeans within a column followed by the same letters are not signicantly dierent (Schees test). cNot tested.

showed that the omission of cuminaldehyde, p-cymene, or -
terpinene from the articial mixture caused a signicant
decrease in the toxicity of the blend (F15,48 = 120.31, p <
0.0001). Cuminaldehyde was the major contributor to the
fumigant toxicity of cumin oil, followed by p-cymene and -
terpinene. This result indicated that cuminaldehyde, p-cymene,
and -terpinene act synergistically in terms of insecticidal
activity against German cockroaches. Jiang et al.21 have already
insisted that plant defense chemicals with more than one mode
of action are especially suitable for plant protection. Omission
of -pinene, -phellandrene, limonene, menthol, and bornyl
acetate did not cause a signicant dierence in the fumigant
toxicity of the blends, but there was a signicant dierence
between the toxicity of the blends and cumin oil. This result
indicated that unidentied compounds (23.42%) must be
responsible for the total toxicity of the oil, and -pinene, -
phellandrene, limonene, menthol, and bornyl acetate might act
more synergistically with the unidentied compounds than -
pinene, -myrcene, cis-linalool oixide, neral, and trans-anethole.
There was no signicant dierence in contact toxicity
between dill, carvi, cumin, and ajowan oils and complete
articial mixtures of these oils (Figure 2). For dill oil,
components elimination assays (Figure 2) demonstrated that
omission of carvone and limonene from the mixture caused a
signicant dierence in the contact toxicity of the blends.
Carvone was also the major contributor for the contact toxicity
of carvi oil (Figure 2). Omission of other compounds did not
cause a signicant dierence in the contact toxicity of the
blends. For cumin oil, several compounds were involved in the
contact toxicity of the oil. Signicant dierence in the contact
toxicity of the blends was observed when cuminaldehyde, p-
7200
cymene, and -terpinene were removed from the complete
mixture. Omission of other compounds did not cause a
signicant dierence in the contact toxicity, but toxicity of the
blends was reduced, in comparison with that of cumin oil or the
complete articial mixture. This result indicated that
unidentied compounds (23.42%) were responsible for the
total contact toxicity of the oil, as mentioned above. Thymol
was the major contributor to the contact toxicity of ajowan oil,
but other compounds did not cause a signicant dierence in
the contact toxicity of the blends (Figure 2).
Primary AChE Inhibition Assay and IC50 Estimation.
The primary inhibition rates of the chemicals identied in the 4
active oils against the German cockroach are summarized in
Figure 3. In males, carvacrol showed the highest inhibition rate
(78%), followed by -pinene (71.6%) (Figure 3). The -pinene
also exhibited >50% inhibition rate (53.4%). However, in
females, -pinene showed the highest inhibition rate (86.3%),
followed by carvacrol and dihydrocarvone (55.1% and 50.6%,
respectively). In the primary inhibition assay, 3 chemicals (-
pinene, carvacrol, and dihydrocarvone) that showed >50%
inhibition rate were selected and their IC50 values against AChE
were estimated. The IC50 of -pinene, carvacrol, and
dihydrocarvone was 0.18, 0.18, and 1.60 mg/mL, respectively,
in males and 0.28, 0.17, and 0.78 mg/mL, respectively, in
females (Table 7). AChE inhibition activity of phytochemicals
has been investigated in several studies.2225 Abdelgaleil et al.26
reported that cuminaldehyde, 1,8-cineole, ()-limonene, and
(L)-fenchone showed strong Sitophilus oryzae AChE inhibition
activity. However, cuminaldehyde, 1,8-cineole, and (+)-limo-
nene showed weak AChE inhibition activity in this study.
Instead, carvacrol, -pinene, and dihydrocarvone demonstrated
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Journal of Agricultural and Food Chemistry Article

Figure 3. Blatella germanica acetylcholine esterase inhibition rates of constituents identied in four active oils: (A) males; (B) females. Mean values
corresponding to each treatment with dierent letters are signicantly dierent from each other (male, F19,40 = 160.57, p < 0.0001; female, F19,40 =
136.28, p < 0.0001, Schees test).

Table 7. Acetylcholine Esterase Inhibition Activity of -Pinene, Carvacrol, and Dihydrocarvone

male female
compounds slope IC50 (mg/mL) 95% cla 2 slope IC50 (mg/mL) 95% cl 2
-pinene 1.01 0.10 0.18 0.130.24 1.22 0.86 9.90 0.28 0.200.38 0.58
carvacrol 1.09 0.10 0.18 0.130.23 6.39 1.08 0.10 0.17 0.120.22 7.61
dihydrocarvone 0.61 0.10 1.60 0.963.65 1.94 0.76 0.10 0.78 0.541.23 2.74
a
Condence limit.

strong AChE inhibition activity. This might be attributed to the
use of dierent insect species. Carvacrol and dihydrocarvone
demonstrated strong fumigant or contact toxicity against
German cockroaches in an individual compound test. Our
results indicated that the toxicity of carvacrol and dihydro-
carvone correlated with the ability to inhibit AChE activity.
Anderson and Coats27 also reported that carvacrol showed
strong reaction for American cockroach AChE inhibition
activity. However, Lei et al.28 insisted that the nematicidal
activity of carvacrol against 2 nematodes, Caenorhabditis elegans
7201
and Ascaris suum, might be mediated through a tyramine
receptor. -Pinene showed strong AChE inhibition activity, but
their fumigant or contact toxicity was weak in comparison with
the toxicity of the other compounds. One possibility for the
weak contact or fumigant toxicity of -pinene could be low
penetration rate to the target site. However, carvacrol, -
pinene, and dihydrocarvone were not major contributors to the
toxicity of the essential oils in the articial blend test. This
nding suggested that the mode of action for the essential oil is
not AChE inhibition. Although little is known regarding the
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Journal of Agricultural and Food Chemistry
mode of action of the essential oils in insects, many oils or their
constituents cause symptoms that indicate a neurotoxic mode
of action.29,30 Eugenol exerts its insecticidal activity by binding
to octopamine receptors.31,32 However, the exact mode of
action of essential oils and constituents tested in this study
remains unclear.
Our results indicate that carvi, dill, cumin, and ajowan oils
and their components could be developed as control agents
against German cockroaches. For the practical use of these oils
and their constituents as novel cockroach-control agents, the
safety of the oils and their components in humans and
nontarget organisms and their mode of action should be
investigated further.
AUTHOR INFORMATION
Corresponding Author
*Tel: +82-2-961-2672. Fax: +82-2-961-2679. E-mail: parkik1@
forest.go.kr.
Notes
The authors declare no competing nancial interest.
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